Field of the Invention
The present invention relates to a device for wetting a sample with an aerosol, preferably a biological sample, for example, a cell sample, in particular at the air-liquid interface. The invention further relates to a corresponding method for wetting a sample with an aerosol. The sample is particularly made up of cells, preferably epithelial cells, most preferably lung cells.
Description of the Background Art
In experiments, it is often necessary to expose a sample, such as cells, to a substance, in particular to a pharmaceutically active substance, e.g. for drug screening, or to a toxic substance, e.g. for toxicity studies.
Usually, in vitro cell experiments are performed for this purpose. Here, an active agent or a toxicologically active substance is added to a sample of cells under so-called submerged cell culture conditions. In such submerged cell culture conditions, the cells are fully covered by the cell medium and the test substance, such as a pharmaceutically active substance or a toxic substance, is pipetted directly into the cell medium.
For epithelial cells, i.e. cells that bound the body to the outside, such as lung epithelial cells, skin cells or retinal cells, this situation is, however, unrealistic because the epithelial cells typically form an air-liquid interface (ALI) in the body. This means that on one side of the epithelial cells there is air or general-purpose gas, and on the other side interstitial tissue or general-purpose liquid.
For studies and experiments on such cells, the complete immersion of such cells in the cell medium is physiologically unrealistic. At the same time, studies or experiments on such cells are often of interest. This is especially true for efficacy and toxicity studies with substances administered by inhalation, for example for inhalation therapies or studies on occupational safety in regards to handling aerosolizable materials, in particular nanomaterials.
Studies at the ALI are used, for example, to analyze the effects of aerosol borne substances (e.g. active agents, toxins, nanoparticles) on cells. What is paramount here is in particular the effect on cells located at the air/bodily fluids contact surface, for example, on epithelial cells of the lung, skin or retina. In inhalation therapy, aerosolized agents are selectively applied to the lung epithelium to treat lung diseases or systemic diseases. In addition, the lung also offers the most important gateway for dealing with many toxic or potentially toxic substances such as environmental dust or nanomaterials in the workplace.
In order to examine those epithelial cells under preferably physiological conditions, several methods have been developed that simulate such an ALI environment. In particular, by means of Transwell inserts, cells at the ALI can also be cultured in standard microtiter plates. Culturing epithelial cells at the ALI is generally physiologically more realistic and therefore, with respect to the effect of substances, potentially more meaningful than the submerged cell cultures described above.
There is therefore a basic need to provide a suitable system for studies and experiments on such ALI cell cultures with which a substance can be evenly applied to ALI cell cultures in a thin layer (e.g. approximately 10-100 microns). Such substances typically include liquid aerosolized agents, but may also include dry substances. Especially for pharmaceutical studies with some very expensive substances, a high deposition efficiency and a uniform distribution of the aerosolized substance on the ALI cells is beneficial. Since neither a suitable device nor a corresponding method have yet been established, the use of ALI cell cultures in pharmaceutical studies and experiments is still relatively limited.
A few systems already exist which aim to coat (lung) cells at the ALI with aerosolized substances. Based on the approach used for the following technological challenges, these substances can be divided into different categories. On the one hand, there is the challenge of transporting the aerosolized substance to the cells (transport) and on the other hand, the object of depositing the aerosol on the cells (deposition).
According to the system in DE102009016364A1, the aerosol is transported to the cells by an air flow (transport: air flow) and deposited by diffusion and/or sedimentation (depending on the size and mass of the aerosol) on the cells.
It is further known to use air flow to transport an aerosol, wherein the deposition occurs electrostatically. Here, the aerosols are initially charged electrostatically and then deposited on the cells electrophoretically in an electric field.
It is also known to apply the cells from a relatively small distance by direct “spraying”. Here, transport takes place by generating high-speed aerosols (for example by means of a high pressure nozzle or a quick carrier air stream) and by depositing on the cells via inertial impaction. These types of systems often have the disadvantage of not being able to guarantee the uniform distribution of the aerosols on the cells, or that the operation of these systems is technically very complex.
Often, the known systems can only be used for toxicologically relevant dry aerosols, but not for pharmaceutical liquid aerosols. In addition, the usable particle sizes are often limited, e.g. to below 1 micron. Larger aerosols, for example, greater than 1 micron, such as those used for inhalation therapy, are often not possible.
Many of the known systems also have a very low substance deposition rate, of, for example, about 0.1 μm/cm2/h or approximately 0.1 nL/cm2/h for aqueous solutions (Paur et al. Journal of Aerosol Science 42 (2011) 668-692, and in here, Table 3), and therefore require very long exposure times of several hours to several days in order to elicit measurable cell-biological responses. Appropriate processes and equipment are very expensive and time-consuming and also make it difficult to work with cells that can be cultured only for 7 days. Operation and quality control also prove to be complex. The (cell) deposition efficiency (percentage of material invested in the aerosol generator which is deposited on the cells) of the known systems is usually far less than 100%, for example, less than 1%, which is sometimes caused by a low deposition efficiency of the aerosols on the cells and by substance losses (residues) in the aerosol generator and in the supply lines. Electrostatic deposition systems also prove to be disadvantageous since the cargo volumes that cannot be avoided could have a distorting effect on the cell response. In addition, some of the known systems are disadvantageous in that they do not allow even or uniform distribution of the substance on the cells. Thus, in particular, no comparable and reproducible dosimetry on the cells is possible. Due to the uncontrolled conditions, a reliable dose-response relationship is difficult to determine. Finally, solutions that deposit the material by inertial impaction are disadvantageous since the cells, among other things, suffer under the high inflow velocity of the aerosol.
Most of the known systems have in common that the aerosol is carried to an exposure chamber via a continuous airflow, requiring a corresponding technological outlay. An example of such a system is the Air-Liquid Interface Cell Exposure system, short ALICE (described in Lenz, A G, E. Karg, B. Lentner, V. Dittrich, C. Brandenberger, B. Rothen-Rutishauser, H. Schulz, G. A. Ferron and O. Schmid, A dose-controlled system for air-liquid interface cell exposure and application to zinc oxide nanoparticles, Particle and Fibre Toxicology 6 (32), 1-17, 2009). An aerosol cloud is transported from one side to an exposure chamber by means of an external air flow. There, the aerosol cloud descends, forms a vortex and then forms a mist that sediments onto the cells, thereby wetting the cells with the substance. The aerosol-depleted air is then removed on the other side of the exposure chamber. This system has a relatively small cell deposition efficiency of approximately 7% and takes up a lot of space (about 1 m3). It can therefore not be operated under a laminar flow cabinet. Furthermore, it has a very complicated technical structure and is therefore more costly to operate. Among other things, the system requires a humidifier, a pump and an air flow meter for generating an external air flow, as well as a droplet trap to avoid disturbances in the exposure process. In addition, the nebulizer is arranged laterally adjacent to the exposure chamber and the system is operated with an external air flow, i.e. in particular, is not operated airflow-free.
Another method which is operated airflow-free was introduced by F. Blank in Blank F, Rothen-Rutishauser B M, Schurch S, Gehr P: An optimized in vitro model of the respiratory tract wall to study particle cell interactions. Journal of Aerosol Medicine-Deposition Clearance and Effects in the Lung, 19(3):392-405, 2006. In this method, a sample is wetted by direct spraying, wherein the sprayer is positioned 12 cm above the sample. The system generates an aerosol spray and deposits the aerosol on the sample via inertial impaction. No sedimentation or cloud effects are used or harnessed. In addition, the system is open (without side walls or cover region) and the method provided herein only achieves a not specified but probably relatively low deposition efficiency. Furthermore, such a spray features a droplet distribution that is heterogeneous at only a very short distance from the production site. Therefore, such a method is not suitable for providing a homogeneous distribution of substances onto samples that are spaced over an area of 100 cm2 or more. Moreover, the reproducibility of the wetting of the sample is not guaranteed because the nebulizer used (MicroSprayer, Penn-Century Inc., USA) is operated manually.